Many purification techniques include a heating step. It is difficult to purify double-stranded DNA using these techniques because heating causes denaturation of double stranded DNA (dsDNA) duplexes to form single-stranded DNA (ssDNA). This causes problems for any downstream DNA processing techniques which require dsDNA.
Various methods for stabilising double-stranded nucleic acids are known. For examples, reference 1 reviews the sequence-specific isolation and purification of intact dsDNA by oligonucleotide/PNA -assisted affinity capture. References 2 & 3 discuss the use of the PD-loop in the isolation of dsDNA fragments from complex mixtures. Reference 4 discloses the use of ion-pair reversed-phase HPLC to purify dsDNA. Other methods are discussed in the introduction to references 5 and 6, and commercially-available kits include the Wizard™ Genomic DNA Purification Kit (Promega), FlexiPrep™ (Pharmacia Biotech) and Whatman BioScience's Genomic DNA Purification System (‘WBPS’; ref. 7).
Reference 5 discloses methods and reagents for stabilising dsDNA. It discloses an aqueous solution for treating a nucleic acid duplex having a pH of from 3 to 11 and comprising (a) a soluble protein or mixture of proteins and (b) 0.1 mM to 10 mM divalent cations. The nature and concentration of the soluble protein or mixture of proteins is selected so that the solution is capable of inhibiting heat denaturation of a nucleic acid duplex.
The preferred proteins used in reference 5 are from mammalian blood serum. By its nature, however, serum is a complex mixture. It would be advantageous to achieve the same effects as disclosed in reference 5 using simpler and/or more defined reagents. It is therefore an object of the invention to provide improved reagents, methods and compositions for use in stabilising nucleic acid duplexes such as dsDNA.